Methods for inhibiting tumor cell proliferation

ABSTRACT

The subject invention concerns methods for inhibition of STAT biological functions using platinum complexes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/481,226, filed Aug. 13, 2003, U.S. Provisional Application Ser.No. 60/515,580, filed Oct. 30, 2003, and U.S. Provisional ApplicationSer. No. 60/525,295, filed Nov. 25, 2003, and U.S. ProvisionalApplication Ser. No. 60/519,943, filed Nov. 14, 2003, the disclosure ofeach of which is incorporated herein by reference in its entirety.

This invention was made with government support under National CancerInstitute grant number 5 P01 CA78038-03. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Cellular responses to growth factors and cytokines are characterized byactivation of the Signal Transducer and Activator of Transcription(STAT) family of cytoplasmic transcription factors (Darnell, 1997;Darnell et al., 1994; Schindler et al., 1995; Stark et al., 1998;Smithgall et al., 2000; Akira, 2000; Hirano et al., 2000; Bromberg etal., 1996; Fukada et al., 1996; Kotenko et al., 2000). STATs areactivated at a very early stage in the transduction pathway by tyrosinephosphorylation that is induced by protein tyrosine kinases of growthfactor receptors, receptor-associated Janus kinase (Jaks) or Src kinasefamilies. This in turn induces phosphotyrosine (pTyr)-SH2 interactionsbetween two STAT monomers and the formation of dimers, which thentranslocate to the nucleus, bind to specific DNA response elements andregulate the expression of genes essential for cell proliferation,differentiation, development and survival.

Normal STAT activation is tightly-regulated and has a short duration,which is in keeping with normal cellular requirements for mounting aresponse to external stimuli. However, persistent activation of specificSTAT proteins, particularly Stat3 and Stat5, occurs with high frequencyin some tumors, and persistently-active Stat3 has a causal role inmalignant transformation by promoting growth and survival of transformedand tumor cells, including those breast, prostate and head and necksquamous carcinoma cells, lymphomas and leukemias (Bromberg et al.,1999; Turkson et al., 1998; Bromberg et al., 1998; Catlett-Falcone etal., 1999a; Garcia et al., 2001; Grandis et al., 2000a; Grandis et al.,1998; Nielsen et al., 1997; Nielsen et al., 1999; Epling-Burnette etal., 2001; reviewed in Bowman et al., 2000a; Turkson et al., 2000; Songet al., 2000; Coffer et al., 2000; Lin et al., 2000; Catlett-Falcone etal., 1999b; Garcia et al., 1998). Of clinical importance, blockade ofStat3 signaling in malignant cells or whole tumors that containpersistently-active Stat3 induces apoptosis and tumor regression.

Platinum complexes, the prototype of cisplatin, have been widely used asactive anticancer agents (Ardizzoni et al., 1999; Nitiss, 2002) in avariety of human tumors, including testicular, ovarian, bladdercarcinoma, head and neck, and non-small cell lung cancers. The outcomeof treatments with cisplatin and other platinum-containing compounds isstrongly linked to their alkylating effects on DNA. However, thepotential impact of platinum-complex-based therapy on cellular signalingand the therapeutic importance of such interactions have yet to beexplored. Reports show that cisplatin induces activation of members ofthe mitogen-activated protein kinase (MAPK) pathways (Persons et al.,1999; Sanchez-Perez et al., 1998), which may influence drug-inducedapoptosis.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns methods for inhibiting STAT proteinfunction using platinum complexes. In one embodiment, a platinum complexis contacted with a cell expressing a STAT protein. The STAT protein canbe, for example, stat3.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Patent Office upon request andpayment of the necessary fee.

FIGS. 1A-D are photographs showing EMSA analyses of DNA-bindingactivities and effects of platinum complexes. Nuclear extractscontaining activated Stat1, Stat3 and Stat5 are treated with theindicated concentrations of platinum complex ISSCPA-1, ISSCPA-3,ISSCPA-7, or PtCl₄ for 30 min at room temperature prior to incubationwith radiolabeled oligonucleotide probes. FIG. 1A shows Stat1 and Stat3binding activities to hSIE probe, FIG. 1B shows Stat1 and Stat5 bindingactivities to MGFe probe, FIG. 1C shows the binding of E2 μl to DHFRsequence as probe, and FIG. 1D shows the binding of NFκB to bindingsequence in alpha-2 macroglobulin promoter as probe. Positions ofcomplexes of DNA with STAT, E2F1 or NFκB (p65, p50) in gel are labeled.Control lanes represent nuclear extracts that are not pre-incubated withplatinum complexes.

FIG. 2 is a photograph showing agarose gel electrophoresis of DNAtreated with or without platinum complexes. Platinum complex-treatedhSIE oligonucleotide and DMSO-treated control oligo were heated to 70°C. for 10 min to unwind, re-annealed by cooling overnight to roomtemperature and analyzed by gel electrophoresis on 2% agarose containingethidium bromide for UV visualization. Position of band corresponding tohSIE in the gel is shown.

FIG. 3 is a photograph showing the evaluation of effects of platinumcomplexes on Stat3 activation analyzed by EMSA. Nuclear extractsprepared from malignant cells that contain constitutively-activatedStat3 and treated with platinum complexes for the indicated times wereanalyzed by EMSA using hSIE oligonucleotide probe. Extracts wereprepared from v-Src-transformed NIH3T3/v-Src; human breast carcinomaMDA-MB-231; MDA-MB-435; and MDA-MB-2468. Position of Stat3 DNA complexin gel is shown.

FIGS. 4A-B show inhibition of Stat3-mediated gene expression in intactcells by platinum complexes. v-Src-transformed mouse fibroblasts thatstably express Stat3-dependent (NIH3T3/v-Src/pLucTKS3) andStat3-independent (NIH3T3/v-Src/pRLSRE) luciferase reporters weretreated with platinum complexes for the indicated times. Cytosolicextracts were then prepared from cells for Stat3-dependent fireflyluciferase activity (FIG. 4A) and Stat3-independent renilla luciferasemeasurements (FIG. 4B). Values are the means and S.D. of fiveindependent assays.

FIGS. 5A-C are photographs showing the effects of platinum complexes onsignal molecules. Fibroblasts over-expressing EGF receptor(NIH3T3/Hegfr) and their v-Src-transformed counterparts (NIH3T3/v-Src)were treated with platinum complexes for 24 h and stimulated with orwithout EGF for 5 min. Whole cell lysates were prepared and analyzed on5% SDSPAGE and transferred to a nitrocellulose membrane and probed byWestern blotting using (Panel A) anti-pTyr antibody (4G10) or antibodiesagainst activated forms of the members of the MAPK family, (Panel B)ERKs, and (Panel C) p38mapk.

FIGS. 6A and 6B show the effects of platinum complexes on cellproliferation and viability. FIG. 6A shows normal or v-Src-transformedmouse fibroblasts in culture treated with platinum complexes for theindicated times and number of viable cells enumerated by visualizationunder microscope and trypan blue exclusion. FIG. 6B shows normal andSrc-transformed mouse fibroblasts, human breast epithelial as well astumor cells of the breast (MDA-MB-231, MDA-MB-435, MDA-MB-453, andMCF-7), lung (A549) and prostate (DU145) treated with or withoutplatinum complexes for 24-48 hrs and analyzed for extent of[³H]thymidine incorporation. Values are the mean and S.D. of 3-4independent determinations.

FIG. 7 is a photograph showing induction of apoptosis by platinumcomplexes. Normal NIH3T3 fibroblasts and their v-Src-transformedcounterparts were treated with platinum complexes for 48 h and analyzedfor evidence of DNA damage using TUNEL staining kit.

FIGS. 8A-C show tumor regression induced by platinum (IV) chloride.Tumor models in mice using B16 melanoma and CT26 colon tumors both ofwhich harbor constitutively-active Stat3 were treated by intra-tumoralinjection with doses of platinum (IV) chloride and tumor sizes monitoredevery other day for up to 9 days. FIG. 8A is a photograph showingextracted tumor tissues were analyzed for Stat3 activity in in vitroDNA-binding assays and EMSA analysis. FIG. 8B and FIG. 8C show tumorsizes monitored by calipers plotted against days of treatment.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence of an oligonucleotide probe.

SEQ ID NO:2 is the nucleotide sequence of an oligonucleotide probe.

SEQ ID NO:3 is the nucleotide sequence of an oligonucleotide probe.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns methods for inhibiting function of STATproteins. Platinum complexes useful in the subject invention are shownbelow:

The subject platinum complexes can be prepared using standard chemicalsynthesis methods and materials known in the art.

Compounds of the subject invention also includepharmaceutically-acceptable salts of the subject platinum complexes. Theterm pharmaceutically-acceptable salts means salts of the platinumcomplexes of the invention which are prepared with acids or bases,depending on the particular substituents found on the subject complexesdescribed herein. Examples of a pharmaceutically-acceptable baseaddition salts include sodium, potassium, calcium, ammonium, ormagnesium salt. Examples of pharmaceutically-acceptable acid additionsalts include hydrochloric, hydrobromic, nitric, phosphoric, carbonic,sulphuric, and organic acids like acetic, propionic, benzoic, succinic,fumaric, mandelic, oxalic, citric, tartaric, maleic, and the like.Pharmaceutically-acceptable salts of platinum complexes of the inventioncan be prepared using conventional techniques.

It will be appreciated by those skilled in the art that certain of theplatinum complexes of the invention may contain one or moreasymmetrically substituted carbon atoms which can give rise tostereoisomers. It is understood that the invention extends to all suchstereoisomers, including enantiomers, and diastereoisomers and mixtures,including racemic mixtures thereof.

The platinum complexes of the subject invention are potent and selectivedisruptors of STAT activity. Exemplified compounds ISSCPA-1 (alsodesignated herein as CPA-1) and ISSCPA-7 (also designated herein asCPA-7) strongly disrupt Stat3 activity and interfere with its ability tobind to its consensus binding sequence. The platinum complexes of theinvention induce cell growth inhibition and apoptosis in transformedcells and tumor cells with persistently active STATs. Malignant cellswith aberrant or constitutive STAT signaling are highly sensitive toplatinum complexes of the invention. General cytotoxicity of the subjectplatinum complexes to normal cells is minimal or nil. The observationthat the exemplified compounds ISSCPA-1 and ISSCPA-7 selectively blockthe growth and replication of transformed and tumor cells that containabnormal Stat3 activity while only slowing the growth of cells that donot have abnormal Stat3 activity is highly significant. In addition,strong apoptosis is induced by platinum compounds of the invention inmalignant cells that harbor persistently-active STAT signaling, whichcorrelates with suppression of aberrant STAT activity in these cells.

Platinum complexes of the subject invention also exhibit anti-tumoractivity in melanoma and colon tumors in vivo. The abrogation ofconstitutively-active STATs in tumors treated with platinum complexes ofthe invention is consistent with their effects on STAT activity both invitro and in whole cells, and together establish STAT-based anti-tumoreffects of these compounds.

Methods of the invention comprise inhibiting function of a STAT bycontacting a cell expressing a STAT with a platinum complex of theinvention wherein the complex is taken in or otherwise provided insidethe cell. In an exemplified embodiment, platinum complexes ISSCPA-1 andISSCPA-7 physically interact with the DNA-binding domain of Stat3 andthereby disrupts its ability to bind to DNA. In Src-transformed mousefibroblasts, as well as in human tumor cells of the breast, prostate,and lung, and mouse melanoma cells that contain constitutive Stat3activity, both ISSCPA-1 and ISSCPA-7 abrogate Stat3 signaling functionand thereby induce cell growth inhibition and apoptosis.

Methods of the invention also comprise inhibiting the function and/orgrowth and replication of a cell that is aberrantly or constitutivelyexpressing a STAT, such as Stat1 or Stat3. In one embodiment, the methodcomprises contacting a cell with a platinum complex of the invention. Inone embodiment, the cell is a tumor cell, cancer cell, or a transformedcell. The cell can be a cell from a mammal, including human, monkey,chimpanzee, ape, dog, cat, horse, cow, or pig. Platinum complexes of theinvention can be delivered to a cell either through direct contact withthe cell or via a carrier means. Carrier means for deliveringcompositions to cells are known in the art and include, for example,encapsulating the platinum complex in a liposome moiety. Another meansfor delivery of a platinum complex of the invention to a cell comprisesattaching the platinum complexes to a protein or nucleic acid that istargeted for delivery to the target cell. Published U.S. PatentApplication Nos. 20030032594 and 20020120100 disclose amino acidsequences that can be coupled to another composition and that allows thecomposition to be translocated across biological membranes. PublishedU.S. Patent Application No. 20020035243 also describes compositions fortransporting biological moieties across cell membranes for intracellulardelivery.

Therapeutic application of the subject platinum complexes, andcompositions containing them, can be accomplished by any suitabletherapeutic method and technique presently or prospectively known tothose skilled in the art. The subject platinum complexes can beadministered by any suitable route known in the art including, forexample, oral, nasal, rectal, and parenteral routes of administration.As used herein, the term parenteral includes subcutaneous, introdermal,intravenous, intramuscular, intraperitoneal, and intrastemaladministration, such as by injection. Administration of the subjectplatinum complexes of the invention can be continuous or at distinctintervals as can be readily determined by a person skilled in the art.An ordinarily skilled clinician can determine an amount of a platinumcomplex of the invention to be administered to a patient that will beeffective to treat the particular condition, disease, or disorder of thepatient.

Compounds useful in the methods of the subject invention can beformulated according to known methods for preparing pharmaceuticallyuseful compositions. Formulations are described in detail in a number ofsources which are well known and readily available to those skilled inthe art. For example, Remington's Pharmaceutical Science by E. W. Martindescribes formulations which can be used in connection with the subjectinvention. In general, the compositions of the subject invention will beformulated such that an effective amount of the bioactive platinumcomplex is combined with a suitable carrier in order to facilitateeffective administration of the composition. The compositions used inthe present methods can also be in a variety of forms. These include,for example, solid, semi-solid, and liquid dosage forms, such astablets, pills, powders, liquid solutions or suspension, suppositories,injectable and infusible solutions, and sprays. The preferred formdepends on the intended mode of administration and therapeuticapplication. The compositions also preferably include conventionalpharmaceutically acceptable carriers and diluents which are known tothose skilled in the art. Examples of carriers or diluents for use withthe subject platinum complexes include ethanol, dimethyl sulfoxide,glycerol, alumina, starch, and equivalent carriers and diluents. Toprovide for the administration of such dosages for the desiredtherapeutic treatment, pharmaceutical compositions of the invention willadvantageously comprise between about 0.1% and 99%, and especially, 1and 15% by weight of the total of one or more of the subject platinumcomplexes based on the weight of the total composition including carrieror diluent.

The compounds of the subject invention can also be administeredutilizing liposome technology, slow release capsules, implantable pumps,and biodegradable containers. These delivery methods can,advantageously, provide a uniform dosage over an extended period oftime. The platinum complexes of the invention can also be administeredin their salt derivative forms or cystalline forms.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Materials and Methods

Cells, plasmids, and other reagents.

v-Src-transformed (NIH3T3/v-Src) and derived counterparts stablyexpressing Stat3 reporter, pLucTKS3 (NIH3T3/v-Src/pLucTKS3) orStat3-independent plasmid, pRLSRE (NIH3T3/v-Src/pRLSRE), andRas-transformed (NIH3T3/v-Ras) fibroblasts, human breast carcinomaMDA-MB-231, MDA-MB-435, MDA-MB-453 and MDA-MB-468, melanoma B16 cells,prostate cancer cells, DU145 and PC3, as well as human lung carcinomaA459 cells have been previously described (Garcia et al., 2001; Turksonet al., 2001; Yu et al., 1995; Johnson et al., 1985). Plasmids pLucTKS3(driving expression of the firefly luciferase gene) and pRLSRE (drivingrenilla luciferase gene (Promega) expression) have been previouslydescribed (Turkson et al., 2001; Turkson et al., 1999). Cells were grownin Dulbecco's modified Eagle's medium (DMEM) containing 5%iron-supplemented bovine calf serum (BCS), with or without G418.

Cytosolic Extract Preparation and Luciferase Assays.

Cytosolic lysates preparation from fibroblasts for luciferase assays orfrom baculovirus-infected Sf-9 insect cells have been previouslydescribed (Turkson et al., 1998; Turkson et al., 2001; Turkson et al.,1999; Zhang et al., 2000). Luciferase assays were performed as outlinedin the supplier's (Promega) manual and measured with a luminometer.

Nuclear Extract Preparation and Gel Shift Assays.

Nuclear extracts were prepared from cell lines and used for EMSA aspreviously described (Turkson et al., 1998; Yu et al., 1995; Garcia etal., 1997). In some cases, cells were pre-treated with platinumcomplexes for the indicated times prior to harvesting. Where cells werestimulated with EGF (6 ng/ml), duration of treatment was 15 min. Nuclearextracts were pre-incubated with compounds for 30 min at roomtemperature prior to incubation with radiolabeled probe. The³²P-radiolabeled oligonucleotide probes used are hSIE (high affinitysis-inducible element, m67 variant, 5′-AGCTTCATTTCCCGTAAATCCCTA-3′) (SEQID NO:1) that binds both Stat1 and Stat3 (Garcia et al., 1997; Wagner etal., 1990), MGFe (mammary gland factor element from the bovine α-caseingene promoter, 5′-AGATTTCTAGGAATTCAA-3′) (SEQ ID NO:2) that binds Stat1and Stat5 (Gouilleux et al., 1995; Seidel et al., 1995), the NF-KBbinding oligo (5′-TCGACAGAGGGGACTTTCCGAGAGGC-3′) (SEQ ID NO:3), and theoligonucleotide sequence from the DHFR promoter that binds E2F1.

Cell Proliferation and TUNEL Staining.

Proliferating cells were counted by phase-contrast microscopy for viablecells (using trypan blue exclusion. TUNEL staining was carried accordingto supplier's instructions to detect apoptotic cells. Cells (NIH3T3 orNIH3T3/v-Src) were first treated with or without compounds 24-48 h priorto staining.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Inhibition of In Vitro Stat3 DNA-Binding Activity by ISSCPAComplexes

Platinum compounds were evaluated for inhibitory activity against STATDNA-binding in vitro. Analysis by EMSA of nuclear extracts prepared fromepidermal growth factor (EGF)-stimulated fibroblast that activatesStat1, Stat3 and Stat5 shows that preincubation (of extracts of equaltotal protein) with different concentrations of ISSCPA-1, ISSCPA-3 (alsodesignated herein as CPA-3), or ISSCPA-7 for 30 min prior to incubationwith ³²P-labeled oligonucleotide, the m67 high affinity sis-inducibleelement (hSIE) probe (hSIE binds Stat1 and Stat3) results indose-dependent reduction in the level of DNA-binding activity of Stat3and Stat1 (FIG. 1A), with compounds twice more potent against Stat3 overStat1 (IC₅₀ values in the low μmolars shown in Table 1 and Table 2). Incontrast similar treatment of nuclear extracts with compounds showedthat they have a much reduced inhibitory effect on Stat5 DNA-bindingactivity (FIG. 1B), suggesting that ISSCPA-1, ISSCPA-3 and ISSCPA-7preferentially disrupt Stat3 and Stat1 activity.

The covalency of platinum in both ISSCPA-1 and ISSCPA-7 is four; thus,whether the observed effects are associated with platinum (IV) wasdetermined. For this purpose, platinum (IV) chloride was evaluated insimilar assay. As analyzed by EMSA, results show that pretreatment ofnuclear extracts with platinum (IV) chloride disrupts DNA-bindingactivity of Stat3 similarly as observed for ISSCPA-1 and ISSCPA-7 (FIG.1A, last 6 lanes). To determine selectivity of compounds for Stat3,effects on other proteins, including E2F1 and NF-KB, were investigated.Analysis by EMSA show that the DNA-binding activities of the twonon-STAT related transcription factors are not significantly altered bycompounds being evaluated (FIGS. 1C and 1D). TABLE 1 IC50 values (μM)for disruption of STAT DNA-binding activity in vitro Stat3:Stat3Stat1:Stat3 Stat1:Stat1 ISS CPA-1 5 9.3 20 ISS CPA-3 5.8 27 8.3 ISSCPA-7 1.5 3.5 4.0

TABLE 2 IC50 values (μM) for inhibition of cell proliferation NIH3T3NIH3T3v-Src ISS CPA-1 — 2.3 ISS CPA-3 — — ISS CPA-7 — 1.3

EXAMPLE 2 Platinum Complexes Do Not Modulate Integrity of DNA Sequence

The ability of Cisplatin and analogs thereof to denature DNA sequencesby alkylation is known. To determine whether platinum complexes of theinvention disrupt DNA integrity, un-annealing and re-annealing studieswere performed with cold hSIE oligonucleotide that have been similarlypre-treated with platinum compounds as done in the in vitro DNA-bindingassay described in Example 1 and re-annealed oligo was analyzed on a1.8% agarose gel and bands visualized by staining the gel with ethidiumbromide. The oligonucleotide sequence treated with ISSCPA-1, ISSCPA-7 orPtCl₄ show an identical migration pattern and band sharpness as thecontrol oligonucleotide that has not been treated with the subjectcompounds (FIG. 2). This finding indicates that treatment of the hSIEoligonucleotide probe with the subject platinum compounds does not havea denaturing effect, suggesting that the subject platinum compounds maynot alter Stat3 binding sequences of the hSIE probe in the in vitroDNA-binding assay.

EXAMPLE 3 Abrogation of Constitutive Stat3 Signaling in Transformed andTumor Cells by ISSCPA-1 and ISSCPA-7

The effects of platinum complexes on persistent activation of Stat3 inSrc-transformed fibroblasts and human tumor cells were investigated. Forcells treated with ISSCPA-1 and ISSCPA-7, EMSA analysis of in vitroStat3 DNA-binding activity in nuclear extracts shows strong inhibitionof constitutive Stat3 activation (FIG. 3), suggesting that the compoundsblock constitutive Stat3 activation. These studies were then extended toevaluate the effects of the platinum complexes on Stat3 transcriptionalactivity. Both ISSCPA-1 and ISSCPA-7 significantly suppressStat3-dependent induction of luciferase reporter (FIG. 4), with littleor no effect on induction of Stat3-independent luciferase activity (FIG.4). In contrast, inhibition of constitutive Stat3 activity by platinumcomplexes ISSCPA-3 and Cisplatin were modest or negligible (FIGS. 3 and4).

To determine whether platinum complexes of the invention have widespreadin vivo effects, changes in other signaling pathways were investigated.The effects of compounds on induction of MAPK, PI-3-kinase and proteinkinase C pathways were evaluated. In Src-transformed NIH3T3/v-Src andNIH3T3/hEGFR stimulated with EGF, treatment with platinum complexes ofthe invention does not alter the levels of tyrosine phosphorylatedproteins as analyzed by Western blotting (FIG. 5A). Similarly, inductionof MAP kinase Erk1 or Erk2 by EGF was not significantly altered bytreatment with compounds (FIG. 5B). These findings together indicatethat ISSCPA-1 and ISSCPA-7 are potent inhibitors of constitutiveactivation of Stat3 in transformed and tumor cells and Stat3-mediatedgene expression.

EXAMPLE 4 Inhibition of Cell Growth and Decreased Cell Viability byPlatinum Complexes

The effects of platinum complexes on cell proliferation measured by[³H]-thymidine incorporation were investigated. Treatment with ISSCPA-1and ISSCPA-7 strongly inhibited the growth of Src-transformed mousefibroblasts, as well as of human breast and non-small cell lung cancercell lines (FIG. 6). In contrast, similar treatment of normal mousefibroblasts or human tumor cells that do not containconstitutively-active Stat3 only has marginal effect, observed as areduction in growth rate of treated cells but not growth inhibitory(FIG. 6). Consistent with a key role for Stat3 in cell growth, this datashows that inhibition of constitutively-active Stat3 induces cell growtharrest.

EXAMPLE 5 Induction of Apoptosis by Platinum Compounds

Studies have established a critical role for constitutive Stat3signaling in the survival of transformed and tumor cells(Catlett-Falcone et al., 1999a; Grandis et al., 2000a; Epling-Burnetteet al., 2001; Bowman et al., 2000b; Grandis et al., 2000b; Horiguchi etal., 2002). Effects of the subject platinum compounds on viability andsurvival of malignant cells were evaluated. To determine whetherinhibition of persistently-active Stat3 by the subject compounds resultsin apoptosis, Src-transformed and normal mouse fibroblasts were treatedwith the subject platinum compounds and subjected to TUNEL staining forevidence of apoptosis. Results in FIG. 7 show that ISSCPA-1 and ISSCPA-7induced strong apoptosis in Src-transformed NIH3T3 fibroblasts. Incontrast, only a marginal degree of apoptosis was observed in normalfibroblasts that were similarly treated. These results demonstrate thatapoptosis is induced in Stat3-dependent transformed cells by platinumcomplexes of the invention.

EXAMPLE 6 Induction of Regression by Platinum (IV) Chloride of Melanomaand Colon Tumors

The antitumor efficacy of platinum (IV) chloride of the invention usingmouse models of melanoma and colon tumors was evaluated. Establishedtumors were directly injected with doses of platinum (IV) chloride andtumor size subsequently monitored. For both the melanoma and colontumors, results show complete regression following treatment withplatinum (IV) chloride (FIG. 8B), while untreated control tumorcontinued to grow. In parallel studies, treated tumors were extractedand Stat3 activity determined on the basis of DNA-binding activityanalyzed by EMSA. In tumors treated with the platinum (IV) chloride ofthe invention, constitutively-active Stat3 was strongly attenuated (FIG.8A). Together, the findings support inhibition of constitutively-activeSTATs by platinum (IV) chloride leads to regression of melanoma andcolon tumors.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

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1. A method for inhibiting a Stat3 transcription factor comprisingcontacting a cell expressing a Stat3 transcription factor with aplatinum complex selected from the group consisting of

or a pharmaceutically-acceptable salt thereof.
 2. The method accordingto claim 1, wherein said cell is a tumor cell, cancer cell, or atransformed cell.
 3. The method according to claim 1, wherein said cellis a mammalian cell.
 4. The method according to claim 3, wherein saidmammalian cell is a human cell, monkey cell, chimpanzee cell, ape cell,dog cell, cat cell, horse cell, cow cell, or pig cell.
 5. The methodaccording to claim 1, wherein said platinum complex is encapsulated in aliposome moiety or said platinum complex comprises a protein or nucleicacid that targets delivery of the platinum complex to said cell.
 6. Themethod according to claim 1, wherein said platinum complex is

or a pharmaceutically-acceptable salt thereof.
 7. The method accordingto claim 1, wherein said platinum complex is

or a pharmaceutically-acceptable salt thereof.